Throughout this application, various publications are referenced within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citations for these references may be found immediately preceding the claims.
HIV-1 entry into target cells is mediated by the successive interaction of the envelope glycoprotein gp120 with CD4 and a co-receptor belonging to the seven transmembrane G protein-coupled chemokine receptor family (Berger et al. Ann. Rev. Immunol. 17:657, 1999). Binding of gp120to CD4 exposes or creates a co-receptor binding site on gp120(Trkola et al. Nature 384:184, 1996, Wu et al. Nature, 384:179, 1996). CCR5 and CXCR4 are the most physiologically relevant and widely used HIV-1 Co- s receptors (Zhang and Moore, J. Virol. 73:3443, 1999). CCRS mediates the entry of RS isolates and CXCR4 mediates the entry of X4 isolates. R5X4 isolates are able to exploit both co-receptors (Berger et al. Ann. Rev. Immunol. 17:657, 1999). It has been demonstrated that specific amino acids including acidic residues and tyrosines located within the CCR5 amino-terminal domain (Nt, amino acids 2-31) are essential for CCR5-mediated fusion and entry of R5 and R5X4 HIV-1 strains (Dragic et al. J. Virol. 72:279, 1998; Rabut et al. J. Virol. 72:3464, 1998; Farzan et al. J. Virol. 72:1160, 1998; Dorantz et al. J. Virol. 71:6305, 1997). More recently, Farzan et al. demonstrated that tyrosine residues in the CCR5 Nt are sulfated (Farzan et al. Cell 96:667, 1999)
Inhibition of cellular sulfation pathways, including tyrosine sulfation, by sodium chlorate decreased the binding of a gp120/CD4 complex to CCR5+ cells (Farzan et al. Cell 96:667, 1999). A number of prior reports had implicated a role for sulfate moieties in HIV-1 entry. Several sulfated compounds, such as dextran sulfate, can inhibit HIV-1 entry by associating with CD4 or gp120 (Baeuerle and Huttner J. Cell Biol 105:2655, 1987; Baba et al. Proc. Natl. Acad. Sci. USA 85:6132, 1998) . Sulfated proteoglycans have been shown to bind to HIV-1 gp120 at or near its third variable (V3) loop, which also determines co-receptor usage (Roderiquez et al. J. Virol. 69:2233, 1995; Hwang et al. Science 253:71, 1991). It is therefore conceivable that sulfotyrosines in the CCR5 Nt also interact with gp120, increasing its affinity for CCR5. The reduction in gp120/CD4 binding caused by the pre-treatment of target cells with sodium chlorate, however, cannot be formally attributed to a reduction in CCR5 tyrosine sulfation since chlorate can inhibit the sulfation of both tyrosines and proteoglycans.
The region of the CCR5 Nt spanning amino acids 2-18 contains residues that are critically important for viral entry (Dragic et al. J. Virol. 72:279, 1998; Rabut et al. J. Virol. 72:3464, 1998; Farzan et al. J. Virol. 72:1160, 1998; Dorantz et al. J. Virol. 71:6305, 1997). We previously demonstrated that tyrosines at positions 3, 10 and 14 were required for optimal co-receptor function, whereas the Tyr15Phe substitution had little effect on entry (Rabut et al. J. Virol. 72:3464, 1998). Taken together, these findings suggested that HIV-1 entry may be critically dependent upon sulfation of Tyr-3, -10 and -14, but not Tyr-15. We therefore explored the role of sulfotyrosines in positions 3, 10 and 14 by synthesizing peptides corresponding to amino acids 2-18 of the CCR5 Nt and carrying different tyrosine modifications. We first tested the ability of the Nt peptides to inhibit binding of gp120/CD4 complexes and anti-CCR5 MAbs to CCR5+ cells. The specific association of certain peptides with gp120/sCD4 complexes or with anti-CCR5 MAbs was further confirmed by surface plasmon resonance (BIAcore) analysis. Inhibition of HIV-1 entry by the CCR5 Nt peptides was also tested. Our results suggest that amino acids 2-18 of the CCR5 Nt compose a gp120-binding site that determines the specificity of the interaction between CCR5 and gp120s from R5 and R5X4 isolates. Post-translational sulfation of the tyrosine residues in the CCR5 Nt is required for gp120 binding and may critically modulate the susceptibility of target cells to HIV-1 infection in vivo.
CCR5""s normal physiologic activities involve binding and transducing signals mediated by CC-chemokines, including RANTES, MIP-1xcex1 and MIP-1xcex2, which direct activation and trafficking of T cells and other inflammatory cells. As such, CCR5 plays an important role in mediating the inflammatory reaction of diseases such as rheumatoid arthritis and multiple sclerosis. The synovial fluid of rheumatoid arthritis patients is highly enriched in CCR5-expressing T cells (Qin et al. J Clin Invest 101:746, 1998), and CCR5 is the predominant CC chemokine receptor expressed on T cells in the rheumatoid synovium (Gomez-Reino et al. Arthritis Rheum 42:989, 1999). Similarly, infiltration by CCR5-expressing cells is characteristic of plaque lesions in patients with multiple schlerosis (Balashov et al. Proc Natl Acad Sci USA 96:6873, 1999). Such observations provide a rationale for the use of agents that block CCR5 for therapy of inflammatory/autoimmune diseases, including but not limited to arthritis, multiple sclerosis, asthma, psoriasis, autoimmune diabetes, transplant rejection, and atherosclerosis.
This invention provides a compound comprising the structure:
xcex8xcex1YDINYYTSExcex2xcex
wherein each T represents a threonine, each S represents a serine, each E represents a glutamic acid, each Y represents a tyrosine; each D represents an aspartic acid, each I represents an isoleucine; and each N represents an asparagine; wherein xcex1 represents from 0 to 9 amino acids, with the proviso that if there are more than 2 amino acids, they are joined by peptide bonds in consecutive order and have a sequence identical to the sequence set forth in SEQ ID NO: 1 beginning with the I at position 9 and extending therefrom in the amino terminal direction; wherein xcex2 represents from 0 to 13 amino acids, with the proviso that if there are more than 2 amino acids, they are joined by peptide bonds in consecutive order and have a sequence identical to the sequence set forth in SEQ ID NO: 1 beginning with the P at position 19 and extending therefrom in the carboxy terminal direction; wherein xcex8 represents an amino group or an acetylated amino group; wherein xcex represents a carboxyl group or an amidated carboxyl group; wherein all of xcex1,Y,D,I,N,Y,Y,T,S,E and xcex2 are joined together by peptide bonds; further provided that at least two tyrosines in the compound are sulfated.
This invention also provides a compound comprising the structure:
xcex8xcex1YDINYYTSExcex2xcex
wherein each T represents a threonine, each S represents a serine, each E represents a glutamic acid, each Y represents a tyrosine; each D represents an aspartic acid, each I represents an isoleucine; and each N represents an asparagine; wherein xcex1 represents from 0 to 9 amino acids, with the proviso that if there are more than 2 amino acids, they are joined by peptide bonds in consecutive order and have a sequence identical to the sequence set forth in SEQ ID NO: 1 beginning with the I at position 9 and extending therefrom in the amino terminal direction; wherein xcex2 represents from 0 to 333 amino acids, with the proviso that if there are more than 2 amino acids, they are joined by peptide bonds in consecutive order and have a sequence identical to the sequence set forth in SEQ ID NO: 1 beginning with the P at position 19 and extending therefrom in the carboxy terminal direction; wherein xcex8 represents an amino group or an acetylated amino group; wherein xcex represents a carboxyl group or an amidated carboxyl group; wherein all of xcex1,Y,D,I,N,Y,Y,T,S,E and xcex2 are joined together by peptide bonds; further provided that at least two tyrosines in the compound are sulfated.
This invention provides a composition which comprises a carrier and an amount of one of the compounds described herein effective to inhibit binding of HIV-1 to a CCR5 receptor on the surface of a CD4+ cell.
This invention provides a method of inhibiting human immunodeficiency virus infection of a CD4+ cell which also carries a CCR5 receptor on its surface which comprises contacting the CD4+ cell with an amount of one of the compounds described herein effective to inhibit binding of human immunodeficiency virus to the CCR5 receptor so as to thereby inhibit human immunodeficiency virus infection of the CD4+ cell.
This invention provides a method of preventing CD4+ cells of a subject from becoming infected with human immunodeficiency virus which comprises administering to the subject an amount of one of the compounds described herein effective to inhibit binding of human immunodeficiency virus to CCR5 receptors on the surface of the CD4+ cells so as to thereby prevent the subject""s CD4+ cells from becoming infected with human immunodeficiency virus.
This invention provides a method of treating a subject whose CD4+ cells are infected with human immunodeficiency virus which comprises administering to the subject an amount of one of the compounds described herein effective to inhibit binding of human immunodeficiency virus to CCR5 receptors on the surface of the subject""s CD4+ cells so as to thereby treat the subject.
This invention provides a method of identifying an agent which inhibits binding of a CCR5 ligand to a CCR5 receptor which comprises:
(a) immobilizing one of the compounds described herein on a solid support;
(b) contacting the immobilized compound from step (a) with sufficient detectable CCR5 ligand to saturate all binding sites for the CCR5 ligand on the immobilized compound under conditions permitting binding of the CCR5 ligand to the immobilized compound so as to form a complex;
(c) removing any unbound CCR5 ligand;
(d) contacting the complex from step (b) with the agent;
and
(e) detecting whether any CCR5 ligand is displaced from the complex, wherein displacement of detectable CCR5 ligand from the complex indicates that the agent binds to the compound so as to thereby identify the agent as one which inhibits binding of the CCR5 ligand to the CCR5 receptor.
This invention provides a method of identifying an agent which inhibits binding of a CCR5 ligand to a CCR5 receptor which comprises:
(a) contacting one of the compounds described herein with sufficient detectable CCR5 ligand to saturate all binding sites for the CCR5 ligand on the compound under conditions permitting binding of the CCRS ligand to the compound so as to form a complex;
(b) removing any unbound CCR5 ligand;
(c) measuring the amount of CCR5 ligand which is bound to the compound in the complex;
(d) contacting the complex from step (a) with the agent so as to displace CCR5 ligand from the complex;
(e) measuring the amount of CCR5 ligand which is bound to the compound in the presence of the agent; and
(f) comparing the amount of CCR5 ligand bound to the compound in step (e) with the amount measured in step (c), wherein a reduced amount measured in step (e) indicates that the agent binds to the compound so as to thereby identify the agent as one which inhibits binding of the CCR5 ligand to the CCR5 receptor.
This invention also provides a method of identifying an agent which inhibits binding of a CCR5 ligand to a CCR5 receptor which comprises:
(a) immobilizing one of the compounds described herein on a solid support;
(b) contacting the immobilized compound from step (a) with the agent and sufficient detectable CCR5 ligand to saturate all binding sites for the CCR5 ligand on the compound under conditions permitting binding of the CCR5 ligand to the immobilized compound so as to form a complex;
(c) removing any unbound CCR5 ligand;
(d) measuring the amount of detectable CCR5 ligand which is bound to the immobilized compound in the complex;
(e) measuring the amount of detectable CCR5 ligand which binds to the immobilized compound in the absence of the agent;
(f) comparing the amount of CCR5 ligand which is bound to the immobilized compound in step (e) with the amount measured in step (d), wherein a reduced amount measured in step (d) indicates that the agent binds to the compound so as to thereby identify the agent as one which inhibits binding of the CCR5 ligand to the CCR5 receptor.
This invention also provides a method of identifying an agent which inhibits binding of a CCR5 ligand to a CCR5 receptor which comprises:
(a) contacting one of the compounds described herein with the agent and sufficient detectable CCR5 ligand to saturate all binding sites for the CCR5 ligand on the compound under conditions permitting binding of the CCR5 ligand to the compound so as to form a complex;
(b) removing any unbound CCR5 ligand;
(c) measuring the amount of detectable CCR5 ligand which is bound to the compound in the complex;
(d) measuring the amount of detectable CCR5 ligand which binds to the compound in the absence of the agent;
(e) comparing the amount of CCR5 ligand which is bound to the compound in step (c) with the amount measured in step (d), wherein a reduced amount measured in step (c) indicates that the agent binds to the compound so as to thereby identify the agent as one which inhibits binding of the CCR5 ligand to the CCR5 receptor.
This invention provides a method of identifying an agent which inhibits binding of a CCR5 ligand to a CCR5 receptor which comprises:
a) immobilizing one of the compounds described herein on a solid support;
b) contacting the immobilized compound from step a) with the agent dissolved or suspended in a known vehicle and measuring the binding signal generated by such contact;
c) contacting the immobilized compound from step a) with the known vehicle in the absence of the compound and measuring the binding signal generated by such contact;
d) comparing the binding signal measured in step b) with the binding signal measured in step c), wherein an increased amount measured in step b) indicates that the agent binds to the compound so as to thereby identify the agent as one which binds to the CCR5 receptor.
This invention provides a method of obtaining a composition which comprises:
(a) identifying a compound which inhibits binding of a CCR5 ligand to a CCR5 receptor according to one of the above methods; and
(b) admixing the compound so identified or a homolog or derivative thereof with a carrier.
This invention provides a compound having the structure:
xcex94xe2x88x92(xcex8xcex1YDINYYTSExcex2xcex)xcfx80
wherein each T represents a threonine, each S represents a serine, each E represents a glutamic acid, each Y represents a tyrosine; each D represents an aspartic acid, each I represents an isoleucine; and each N represents an asparagine; wherein xcex1 represents from 0 to 9 amino acids, with the proviso that if there are more than 2 amino acids, they are joined together by peptide bonds in consecutive order and have a sequence identical to the sequence set forth in SEQ ID NO: 1 beginning with the I at position 9 and extending therefrom in the amino terminal direction; wherein xcex2 represents from 0 to 13 amino acids, with the proviso that if there are more than 2 amino acids, they are joined together by peptide bonds in consecutive order and have a sequence identical to the sequence set forth in SEQ ID NO: 1 beginning with the P at position 19 and extending therefrom in the carboxy terminal direction; wherein xcex represents a carboxyl group or an amidated carboxyl group; wherein all of xcex1,Y,D,I,N,Y,Y,T,S,E and xcex2 are joined together by peptide bonds, further provided that at least two tyrosines in the compound are sulfated, wherein xcfx80 is an integer from 1 to 8, xcex94 is a polymer, and the solid line represents up to 8 linkers which attach the structure in parentheses to xcex94.
This invention also provides a compound having the structure:
(xcex8xcex1YDINYYTSExcex2)xcfx80xe2x88x92xcex94
wherein each T represents a threonine, each S represents a erine, each E represents a glutamic acid, each Y epresents a tyrosine; each D represents an aspartic acid, each I represents an isoleucine; and each N represents an asparagine; wherein xcex1 represents from 0 to 9 amino acids, with the proviso that if there are more than 2 amino acids, they are joined together by peptide bonds in consecutive order and have a sequence identical to the sequence set forth in SEQ ID NO: 1 beginning with the I at position 9 and extending therefrom in the amino terminal direction; wherein xcex2 represents from 0 to 13 amino acids, with the proviso that if there are more than 2 amino acids, they are joined together by peptide bonds in consecutive order and have a sequence identical to the sequence set forth in SEQ ID NO: 1 beginning with the P at position 19 and extending therefrom in the carboxy terminal direction; wherein xcex8 represents an amino group or an acetylated amino group; wherein all of xcex1,Y,D,I,N,Y,Y,T,S,E and xcex2 are joined together by peptide bonds, further provided that at least two tyrosines in the compound are sulfated, wherein xcfx80 is an integer from 1 to 8, xcex94 is a polymer, and the solid line represents up to 8 linkers which attach the structure in parentheses to xcex94.
This invention provides a compound having the structure:
xcex94xe2x88x92(xcex1YDINYYTSExcex2xcex)xcfx80
wherein each T represents a threonine, each S represents a serine, each E represents a glutamic acid, each Y represents a tyrosine; each D represents an aspartic acid, each I represents an isoleucine; and each N represents an asparagine; wherein xcex1 represents from 0 to 9 amino acids, with the proviso that if there are more than 2 amino acids, they are joined together by peptide bonds in consecutive order and have a sequence identical to the sequence set forth in SEQ ID NO: 1 beginning with the I at position 9 and extending therefrom in the amino terminal direction; wherein xcex2 represents from 0 to 333 amino acids, with the proviso that if there are more than 2 amino acids, they are joined together by peptide bonds in consecutive order and have a sequence identical to the sequence set forth in SEQ ID NO: 1 beginning with the P at position 19 and extending therefrom in the carboxy terminal direction; wherein xcex represents a carboxyl group or an amidated carboxyl group; wherein all of xcex1,Y,D,I,N,Y,Y,T,S,E and xcex2 are joined together by peptide bonds,
further provided that at least two tyrosines in the compound are sulfated, wherein xcfx80 is an integer from 1 to 8, xcex94 is a polymer, and the solid line represents up to 8 linkers which attach the structure in parentheses to xcex94.
This invention also provides a compound having the structure:
(xcex8xcex1YDINYYTSExcex2)xcfx80xe2x88x92xcex94
wherein each T represents a threonine, each S represents a 30 serine, each E represents a glutamic acid, each Y represents a tyrosine; each D represents an aspartic acid, each I represents an isoleucine; and each N represents an asparagine; wherein xcex1 represents from 0 to 9 amino acids, with the proviso that if there are more than 2 amino acids, they are joined together by peptide bonds in consecutive order and have a sequence identical to the sequence set forth in SEQ ID NO: 1 beginning with the I at position 9 and extending therefrom in the amino terminal direction; wherein xcex2 represents from 0 to 333 amino acids, with the proviso that if there are more than 2 amino acids, they are joined together by peptide bonds in consecutive order and have a sequence identical to the sequence set forth in SEQ ID NO: 1 beginning with the P at position 19 and extending therefrom in the carboxy terminal direction; wherein xcex8 represents an amino group or an acetylated amino group; wherein all of xcex1,Y,D,I,N,Y,Y,T,S,E and xcex2 are joined together by peptide bonds, further provided that at least two tyrosines in the compound are sulfated, wherein xcfx80 is an integer from 1 to 8, xcex94 is a polymer, and the solid line represents up to 8 linkers which attach the structure in parentheses to xcex94.